Arquipelago - Life and Marine Sciences ISSN: 0873-4704

Living inside termites: an overview of symbiotic interactions, with emphasis on flagellate protists

SÓNIA DUARTE, L. NUNES, P.A.V. BORGES, C.G. FOSSDAL & T. NOBRE

Duarte, S., L. Nunes, P.A.V. Borges, C.G. Fossdal & T. Nobre 2017. Living inside termites: an overview of symbiotic interactions, with emphasis on flagellate protists. Arquipelago. Life and Marine Sciences 34: 21-43.

To degrade lignocellulose efficiently, lower termites rely on their digestive tract’s specific features (i.e., physiological properties and enzymes) and on the network of symbiotic fauna harboured in their hindgut. This complex ecosystem, has different levels of symbiosis, and is a result of diverse co-evolutionary events and the singular social behaviour of termites. The partnership between termites and flagellate protists, together with prokaryotes, has been very successful because of their co-adaptative ability and efficacy in resolving the needs of the involved organisms: this tripartite symbiosis may have reached a physiologically stable, though dynamic, evolutionary equilibrium. The diversity of flagellate protists fauna associated with lower termites could be explained by a division of labour to accomplish the intricate process of lignocellulose digestion, and the ability to disrupt this function has potential use for termite control. Multi-level symbiosis strategy processes, or the cellulolytic capacity of flagellate protists, may lead to innovative pathways for other research areas with potential spin-offs for industrial and commercial use.

Key words: flagellate protists, hindgut symbiotic fauna, lignocellulose digestion, subterranean termites

Sónia Duarte1,2 (email:[email protected]), Lina Nunes1,2, Paulo A.V. Borges2, Carl G. Fossdal3 & Tânia Nobre4, 1LNEC, National Laboratory for Civil Engineering, Structures Department, Av. do Brasil, 101, PT-1700-066, Lisbon, Portugal, 2cE3c, Centre for Ecology, Evolution and Environmental Changes / Azorean Biodiversity Group and University of the Azores, Faculty of Agrarian and Environmental Sciences, PT- 9700–042 Angra do Heroísmo, 3NIBIO Norwegian Institute for Bioeconomy Research, Division for Biotechnology and Plant Health, Forest Health, NO-1431 Ås, Norway, ICAAM – Institute of Mediterranean Agricultural and Environmental Sciences, Laboratory of Entomology, University of Évora, Évora, Portugal.

INTRODUCTION areas of high diversity are located in the tropics, particularly in Africa, South America and Asia Termites are social insects closely related to (Krishna et al. 2013). Termites can be informally cockroaches, from which they evolved (Lo et al. divided into two groups: lower (all families but 2000; Inward et al. 2007a; Engel et al. 2009) and Termitidae) and higher termites (Termitidae), are denominated as Polyneoptera a monophyletic based on the presence or absence of flagellate group including the cockroaches, Dermaptera, protists in their hindgut, respectively, and also on Plecoptera, Orthoptera, Embioptera,Phasmatodea, different feeding and nesting habits, and different Mantophasmatodea, Grylloblattodea, Mantodea, intestinal compartmentalisation (Eggleton 2011; and Zoraptera. More than 3,000 species of Hongoh 2011; Krishna et al. 2013). These insects termites have been described globally, but their are abundant in many terrestrial ecosystems,

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Duarte et al. particularly in the tropics where they are a retained their bacterial symbionts, but lack the dominant invertebrate group that heavily protozoan gut symbionts. They have various contributes to the lignocellulose decomposition feeding habits, with clear separation of feeding process – thus have been called ‘ecosystem and nesting sites, and exhibit a highly engineers’ (Eggleton 2011; Palin et al. 2011). compartmentalised intestine (except for Termites also have a major role in diverse Macrotermitinae and Sphaerotermitinae). ecosystem functions, such as nutrients and Dissimilarly, lower termites feed strictly on organic matter cycling and redistribution, soil lignocellulose and are aided by hindgut symbionts fertility promotion, generation and regulation of during the digestion process; they are considered soil biodiversity and ecosystem restoration to be an ancestral branch of termites which comprises 11 families (Krishna et al. 2013). (Zimmermann et al. 1982; Bignell & Eggleton Lower termites have a dilated section of the 2000; Sugimoto et al. 2000; Jouquet et al. 2011). anterior hindgut (the paunch) where the bulk of They are major contributors to the ecologic symbiotic microbiota is harboured. Most lower stability of their habitats. By preparing different termites nest and feed within the same wood substrates, like wood or leaf-litter, into forms resource. With potential impact on within-nest easily accessed by microorganisms, termites play endosymbiont transmission, lower termites rely a major role as ecosystem conditioners (Lawton on regurgitation of crop contents and saliva et al. 1996). Termites are able to degrade (stomodeal trophallaxis) as well as proctodeal lignocellulose efficiently (e.g. Ohkuma 2008; trophallaxis, involving anus-to-mouth exchanges Husseneder 2010; Watanabe & Tokuda 2010) and of hindgut fluids, to pass food and gut contents to their feeding habits span a gradient from sound nest mates, whereas higher termites rely mainly wood to other lignocellulosic plant materials with on stomodeal trophallaxis (Eggleton 2011; different humification gradients, such as plant Shimada et al. 2013; Mirabito & Rosengaus litter or soil (Sleaford et al. 1996). This niche 2016). Proctodeal trophallaxis fosters the social, differentiation has allowed termites to promote an nutritional and symbiotic fauna interactions impact on the global terrestrial carbon cycle, among lower termites belonging to the same exceeding the cumulative decomposition roles of colony, probably playing a key role in the other arthropods (Bignell et al. 1997). integration of the information of these different Efficient lignocellulose utilisation as a food environments (Nalepa 2015). Trophallaxis may source by termites is possible because of the be horizontal, among nestmates, or vertical, establishment of endo- and ectosymbiosis, among parents and offspring. including microorganisms of all major taxa. These symbionts are Archaea and Bacteria, and LOWER TERMITES GLOBAL IMPACT protists: unicellular eukaryotes belonging to two Because of their feeding habits and preferences, separate lineages, the parabasalids and the lower termites have an important ecological oxymonads (Fig.1) (Bignell 2000; Bignell & impact on diverse ecosystems, but are also Eggleton 2000; Brune & Ohkuma 2011; Adl et al. considered to be structural, agricultural and 2012; Brune 2013). Ectosymbiosis has evolved in forestry pests, as they attack cultivated plants and the fungus-growing termites (Macrotermitinae) forest nurseries (Rouland-Lefèvre 2011). Lower which cultivate a basidiomycete fungus termites account for 80% of the economically (Termitomyces spp.) (e.g. Nobre & Aanen 2012), important species known to cause major problems whereas the majority of the other termites rely in artificial constructions (Nobre & Nunes 2007; solely on endosymbiosis. The fauna harboured Rust & Su 2012). There is concern that the inside the hindgut assists the termite host with number of invasive termite species has increased energy metabolism, nitrogen and vitamin supply more than 50% since 1969 (Evans et al. 2013), and also additional defence mechanisms (Salem which may be related to the globalisation of trade. et al. 2014, Peterson et al. 2015, Zheng et al. In 2010, the global economic impact of termites was 2015). estimated at 35.6 billion euros, and subterranean Higher termites, account for nearly 75% of termites accounted for 80% of this figure, i.e. Isoptera species richness and yet belong to a approximately 24 billion euros (Rust & Su 2012). single family, Termitidae. These termites have 22

Living inside termites

– Lower termites; FGT FGT termites; Lower – is with basidiomycete fungi; than one feeding group. ovan et al. 2001, Eggleton & Tayasu 2001, Inward groups groups of termites (LWT -feeding habits. Some subfamilies have representatives in more more in representatives have subfamilies Some habits. -feeding gut gut through the establishment of a mutualistic external symbios

feeding termites) based on their symbiotic fauna [based on: Don on: [based fauna symbiotic on their based termites) feeding ition ition of externalised mmon mmon ancestor of the cockroach type, of the simplified feeding Soil- – ists; D=acquisition of strict soil strict of D=acquisition ists; Other higher termites; SFT – -growing termites; OHT

23 Fig. Fig. 1. Possible evolutionary trajectory, from a most recent co Fungus et al. 2007b, Bujang et al. 2014; Otani et al. 2014]. A= acquis prot flagellate of C=loss protists; flagellate of B=acquisition

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As the human population increases, production, LIGNOCELLULOSE DIGESTION trade and use of wooden structures and bio- products susceptible to termite infestation FEEDING SUBSTRATE increases, potentially increasing the spread of Wood is a natural material composed of three termite pest species. Warmer seasons and changes main types of components: cellulose (framework in precipitation patterns due to climate change are substance), hemicellulose (matrix substance expected to influence termite territory size and present between cellulose microfibrils) and lignin distribution. For example, the known 27 species (incrusting substance for cell wall solidification). of invasive termite species are likely to increase For the digestion of these main components, their ranges (Su & Scheffrahn 2000; Evans et al. several enzymes are needed; some of those 2013), favouring termite populations in places enzymes are not yet identified in lower termites, where their presence was previously limited by such as exoglucanases or hemicellulases, and these factors (Lal 2004; Peterson 2010; Lee & enzymes present inside the flagellate protists, Chon 2011; Guerreiro et al. 2014). There is thus a enabling the provision of these cellulases during need to develop efficient preventive and control lignocellulose digestion (Hongoh 2011). methods for avoiding possible future termite Cellulases are enzymes which have the ability to pests’ outbreaks. produce sugars from crystalline cellulose (Slaytor Subterranean termite control strategies are 1992). Cellulose and hemicellulose are thus studied and applied worldwide and mainly rely on degraded to sugars, which are then processed into the use of chemical or physical barriers, wood acetate, hydrogen and carbon dioxide. These treatment with insecticides or wood modification products may be used directly by the termite or by acetylation, furfurylation or other techniques, may interact with other nutrients processed inside and subterranean termite population control using termite hindgut. Acetate is used as the main baits. Few of the primary issues in termite control energy source of the termite (Breznak 1982). are the need to efficiently kill the entire colony and the durable protection of the materials. DIGESTIVE TRACT Difficulties arise due to the cryptic and diffuse Termites’ ability to efficiently digest cellulose nature of rhinotermitid pest termite species, relies not only on chemical features (cellulolytic which forage either in extensive underground enzymes), but also on the digestive tract’s galleries, build nests hidden underneath the soil physiological properties. The termite digestive surface, or live in small colonies inside the wood tract is composed of different parts: mouth, they infest. Subterranean termite control has salivary glands, foregut, midgut and hindgut (Fig. relied on the use of persistent, broad spectrum 2; salivary glands not shown in this figure), and insecticides applied to the soil beneath structures. each part has a specific function in terms of Therefore, in the last few decades, although much lignocellulose breakdown. The lignocellulose remains to be investigated, integrated pest breakdown starts in the mouth, with the use of management strategies (IPM) have been solid and hardened mandibles to chew the favoured, such as local spot treatment of infested wooden substrates; the crop and the timber and population control through the use of proventriculus are additional organs situated in baits and insect growth regulators (like chitin the foregut, which are responsible for further synthesis inhibitors) (Evans & Iqbal 2014). milling and filtering of the ingested wood If the biology and ecology of the pest species particles (Watanabe & Tokuda 2010; Brune & are considered, management strategies can Ohkuma 2011). Indeed this physical conditioning potentially become more specific, and therefore of the food is crucial for efficient digestion as it also potentially more sustainable. Further results in proper cleavage of the substrate and understanding of how termites feed and obtain thus facilitates the access of cellulolytic enzymes nutrients and grow as a colony will assist in developing greatly needed innovative termitecontrol methods.

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The hindgut harbours a rich symbiotic fauna, years of research. Only after Cleveland (1923) was which was thought to be parasitic in the early clear that termites devoid of hindgut symbionts

Fig. 2. Photo showing a worker and the extracted gut with different parts of the subterranean termite Reticulitermes grassei Clément gut: Foregut; Midgut, including the Malpighian tubules (MP) at the posterior end of the midgut; Hindgut were unable to digest lignocelluloses fully, al. 1996; Inoue et al. 1997; Scharf et al. 2011a; suggesting that the relation between host termite Xie et al. 2012; Tsukagoshi et al. 2014). and the hindgut fauna was nutritional symbiosis. The enlarged hindgut is a key structure for Since then, the evidence of flagellate protists’ lower termites’ ability to digest lignocellulose contribution to lignocellulose digestion, through efficiently, as it concentrates major chemical their cellulolytic enzymes, has been demonstrated action on cellulose, with a dilated paunch and is widely accepted (e.g. Yamin & Trager harbouring the majority of symbiotic fauna. 1979; Yamin 1980; Slaytor 1992; Yoshimura et Though fairly simple in lower termites, the anterior and posterior paunch, and the anterior

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Duarte et al. and posterior colon are sequentially structured for MULTI-LEVEL SYMBIOSIS digestion, each serving as defined micro-niches in terms of gradients of oxygen, hydrogen and pH, TERMITE AND ITS SYMBIOTIC FAUNA AS A HOLOBIONT created by a combination of host and hindgut Symbiosis was defined by Anton de Bary in 1879 symbiont activities (Brune & Friedrich 2000), as the ‘living together of different species’; creating difficulties for researchers to reproduce however, this definition does not describe the the physical-chemical conditions of the hindgut complex nature of the relation between termites within the laboratory. Radial concentrations of and their gut symbiotic fauna. The highly oxygen and hydrogen showed a peripheral zone complex ecosystem with different levels of of the hindgut where oxygen is available, and symbiosis inside termite guts is the result of enabling the survival of both aerobic or complex and diverse evolutionary events and also facultative aerobic microorganisms, whereas in of the singular social behaviour of termites. This the hindgut centre, an anaerobic environment is relationship certainly goes beyond the Anton de established, with a zone with high hydrogen Bary concept of symbiosis. Besides the hindgut concentration, resulting from the activity of symbiotic microbiota, the termite colony has also flagellate protists, which release hydrogen, been considered as an organism, since the basic carbon dioxide and acetate and are anaerobic functions are clearly divided in its different parts: (Brune et al. 1995). The hydrogen and carbon reproduction and dispersion (queens, kings and dioxide produced is then used by prokaryotes for alates), construction, feeding and tending methanogenesis or acetogenesis, whereas acetate (workers), active defence (soldiers) and may be used by the termite host (Brune 1998, protection, homoeostasis and fortification (nest) 2013). Furthermore, to harbour anaerobic (Eggleton 2011). The gut symbiotic fauna may be flagellate protists, the termite hindgut functions as directly involved not only in feeding functions, an oxygen sink and this gas needs to be consumed but also in tending, defence, and homoeostasis (Brune et al. 1995). Therefore, flagellate protists (Matsuura 2001; Ugelvig & Cremer 2012; are often associated with methanogenic bacteria Chouvenc et al. 2013; Sen et al. 2015). The and other facultative or strict aerobic hindgut symbiotic fauna is probably also involved in microbiota which consume the oxygen, shaping the termite social behaviour. For maintaining an anoxic environment in some parts example, recently it was shown that lower of the paunch (Brune 1998; Brune & Friedrich termites exhibit different undertaking behaviour 2000). towards conspecific (necrophagy) or congeneric Additionally, endogenous cellulases in lower (burial behaviour) termite corpses. This behaviour termites have been identified and are harboured was interpreted as a defence mechanism together mainly in the salivary glands, playing an with a cost mitigation strategy (Sun et al. 2013). important role in the lignocellulose degradation Another possible advantage of this behaviour may process (Slaytor 1992; Watanabe et al. 1998; be the protection of the hindgut symbiotic fauna, Scharf & Tartar 2008; Scharf et al. 2011a; König avoiding the introduction of new elements into et al. 2013; Peterson et al. 2015). The the hindgut fauna by the ingestion of corpses of lignocellulose digestion is probably the result of termites belonging to other species. The term synergistic action of both termites and their holobiont has been accepted to refer to the host symbionts; though the degree of nutritional and its microbiota as a whole unit able to live, mutualism has some times been questioned develop, survive and evolve together (Rosenberg (Scharf et al. 2011a; Scharf 2015). Recent & Zilber-Rosenberg 2013; Scharf 2015). The metatranscryptomic analysis of termite hindgut cooperation between hosts and their microbiota content has indeed confirmed that the results in a positive contribution to the fitness of lignocellulolytic system has a tripartite origin: the association, providing an increased ability to protists, bacteria and termites (Xie et al. 2012; adapt more rapidly to changing conditions Peterson et al. 2015). (Rosenberg & Zilber-Rosenberg 2013). The

capacity to tackle imposed changes and stresses

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Living inside termites results from the synergy of combined capacities. trophallaxis. This within-nest symbiont Furthermore, social insects such as termites are transmission was observed in a genomic and robust in resisting genetic diversity losses during metagenomic study of a fungus-growing termite phenomena such as introductions or other sources (Poulsen et al. 2014). The obligatory vertical of low population genetic bottlenecks (Ugelvig & mode of transmission of the gut symbiotic fauna Cremer 2012). From this perspective, it is clear to the next generation probably determines the that fitness changes in the termite colony can be gut symbiotic structure associated with termite induced at different levels. species colonies, leading thus to higher levels of Unarguably, the symbiotic association between host-symbiont specificity (Hongoh et al. 2005; lower termites and their hindgut symbionts has Noda et al. 2007; Husseneder 2010). advantages for both, since the termites are able to Some evidences of co-evolution between receive contributions to their main energy supply flagellate protists and their host termites rely on resulting from lignocellulose digestion, and phylogenetic analyses which show a clear co- hindgut symbionts have shelter, protection and diversification pattern, although factors such as food, supplied by the termite host (Radek 1999; stochastic, dietary and ecological effects are also Noda et al. 2009; Brune 2013; Tamschick & important in the long term evolution of symbiont Radek 2013). Probably, the nature of the communities (Tai et al. 2015). Parabasalid symbiotic fauna, and the relationships between protists seem to be strongly influenced by host and amongst them, are driven by host social phylogeny, and the symbiotic bacteria behaviours and shifts in feeding habits. However, communities seem to be more influenced by some have argued that the symbiotic microbes dispersal and environmental acquisition (Tai et al. lead to the development of social habits in wood- 2015). Within the parabasalids, the feeding insects (termites and cockroaches) (e.g. Cristamonadea class seems to be strongly Nalepa et al. 2001), so the paradox remains. associated with the Kalotermitidae family, Nevertheless, symbionts may impose important whereas the Spirotrichonymphea class is linked selective pressures on their hosts, as well as diet with the Rhinotermitidae termite family (Tai et al. preferences or feeding habits (Rosengaus et al. 2015). 2011; Xiang et al. 2012; King et al. 2013). In contrast, flagellate protists belonging to the The tripartite symbiosis between termite host, genus Trichonympha are widely distributed and flagellate protists, and prokaryotes responsible for were detected in six different termite families and lignocellulose degradation translates into a unique also inside the wood-feeding cockroaches symbiotic community, which is probably under belonging to the genus Cryptocercus. This strong co-evolutionary pressure. Indeed, the supports the hypothesis that Trichonympha spp. majority of gut microbiota are considered to be symbionts were acquired by the most recent autochthonous symbionts likely to have co- common ancestor of termites and wood-feeding evolved with their termite host species (Hongoh Cryptocercus spp. cockroaches (Inward et al. et al. 2005; Noda et al. 2007; Tai et al. 2015). The 2007a; Carpenter et al. 2009; Ikeda-Ohtsubo & invasion of new habitats or the proximity of Brune 2009; Tai et al. 2015). It is important to different termite species does not seem to highlight, however, that some authors found a influence the gut symbiotic fauna structure of greater cryptic diversity of Trichonympha species termite species, which tends to maintain its inside one host, using molecular analyses, than integrity in terms of diversity of symbiont species the diversity predicted by use of morphological (Kitade 2004; Hongoh et al. 2005; Husseneder et analyses only (James et al. 2012; Tai et al. 2013). al. 2010; Boucias et al. 2013). Within a termite This suggests that termite hindgut diversity colony, a rather species-specific gut symbiotic estimations could be biased because of the fauna is expected, as termites rely on horizontal underestimation of symbiont diversity, especially transmission of hindgut symbionts to recover the considering that Trichonympha spp. are among hindgut symbiotic community, since, when they the largest species of flagellate protists living moult, symbionts are also discarded. The inside termites (James et al. 2012; Tai et al. recovery is done by proctodeal or stomodeal 2013). 27

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Intracellular bacterial symbionts of termite 2013). The termite hindgut has proven to be a rich hindgut flagellates belonging to Endomicrobia source of novel organisms, including new (phylum Termite Group 1) tend to form a unique findings for science at high taxonomic levels phylogenetic lineage with either their flagellate (Brune 1998; Ohkuma 2003; Boucias et al. 2013, protist host species or their termite host, Sato et al. 2014; Tai et al. 2015). suggesting co-speciation events (Ikeda-Ohtsubo The maintenance of an anoxic environment in et al. 2007; Tai et al. 2015). Co-speciation some parts of the paunch is thought to be just one between Bacteroidales endosymbionts and their of the many putative roles of the symbiotic host protists was demonstrated in 13 out of 14 bacteria. These bacteria exhibit different levels of taxa of the protist genus Pseudotrichonympha, association: free-living in the hindgut (symbiotic and in turn these protist species showed an almost with the insect host), directly associated with complete co-speciation with host termite species flagellate protists (either as endo- or as belonging to the Rhinotermitidae (Noda et al. ectosymbionts) or associated with the gut wall 2007). In a recent study of the ‘Endomicrobia’ (Tamschick & Radek 2013). The tasks of associated with Trichonympha genus, strict host symbiotic bacteria (prokaryotes) and their roles specificity was concluded (Zheng et al. 2015). In are not completely understood and many species contrast, oxymonads’ and parabasalids’ remain undescribed (for a review on this issue see ‘Endomicrobia’ symbionts are rather similar Brune 2013). Brune (2013) defined different between flagellate protist hosts, suggesting that types of hindgut prokaryotes including: they are horizontally transmitted among different lignocellulolytic bacteria, bacteria involved in flagellate protist species living inside the same oxygen reduction reactions, fermentation bacteria, termite hindgut, accounting for the high levels of bacteria responsible for hydrogen metabolism and symbiont transfer between flagellate protist hosts nitrogen-fixing bacteria. and thus lacking (or having reduced levels) of Recently, cultivation-independent studies host-symbiont specificity (Ikeda-Ohtsubo et al. helped to identify and catalogue hindgut 2007; Ikeda-Ohtsubo & Brune 2009). prokaryotes in relation to their function inside the An obligatory ectosymbiont of the flagellate termite hindgut, including some evidences on protist Devescovina spp. has been identified, their role in lignocellulose digestion (Warnecke et suggesting that this highly specific relationship al. 2007; Boucias et al. 2013; He et al. 2013; King evolved as a consequence of strong metabolic et al. 2013; Peterson et al. 2015). Advances in interactions (Desai et al. 2010). sequencing and its accessibility raise expectations At another level of this symbiotic network, the regarding future unveiling of the role of bacteria adaptation of morphological characters of both inside the termite hindgut. host protists and ecto- and endosymbiotic bacteria has been observed (Noda et al. 2007, 2009). FLAGELLATE PROTISTS Specialised cell-surface features which facilitate Flagellate protists are part of the unicellular bacterial attachment indicate a close integration eukaryotes belonging to two separate lineages: of these ectosymbionts in the protist metabolism, the order Oxymonadida (Phylum Preaxostyla) and corroborate the co-evolution hypothesis on and the Phylum Parabasalia (Brugerolle 1991; their symbiotic relationships (Radek et al. 1992; Moriya et al. 1998; Čepička et al. 2010; Adl et al. Dolan et al. 2000). 2012). In spite of the difficulties in laboratory cultivation of most of these organisms, their HINDGUT PROKARYOTES taxonomy was initially based on morphological Most of the hindgut prokaryotes are bacteria, characters (e.g. Brugerolle 1991; Cavalier-Smith whereas the Archaea represent a low percentage 1993). With culture-independent techniques it has of hindgut symbionts. The major groups of become possible to reconstruct the phylogeny of bacteria usually identified in lower termite some groups of flagellate protists, and some hindgut are: Spirochaetes, Bacteroidetes, studies are based on both morphological and Elusimicrobia and Firmicutes (Clostridiales) molecular data (e.g. Carpenter et al. 2010; (Stingl et al. 2005; Boucias et al. 2013; Brune Čepička et al. 2010).

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The oxymonads do not have energy-generating Protists represent the majority of termite hindgut organelles or a highly developed intracellular microorganisms (Katzin & Kirby 1939; Inoue et membrane systems, like dictyosomes, and live al. 1997; König et al. 2013). The general mostly inside insects. Oxymonads may be motile uniqueness of these protists to termites and to or attached to termite hindgut walls. The most wood-feeding cockroaches belonging to the genus common oxymonads living inside termite hindgut Cryptocercus highlights the origin of termites belong to the genera Dinenympha, Pyrsonympha from a cockroach-like ancestor, corroborated by or Oxymonas (Brugerolle & Radek 2006). In molecular phylogenetic data (Inward et al. contrast to the oxymonads, the parabasalids have 2007a). The mode of transmission of protists and anaerobic energy-producing organelles the close relationships developed between host (hydrogenosomes) and a characteristic parabasal and symbionts in terms of metabolic interactions apparatus, which is comprised of dictyosomes and needs probably account for the developed associated with parabasal fibres. Parabasalids specificity (Kitade et al. 2012). Based on recent may be large and are highly motile, with four or attempts to infer termite family relationships more flagella. These protists live mostly inside (Engel et al. 2009; Bourguignon et al. 2015), and termites and cockroaches as symbionts, but some according to available data on flagellate protist species may be parasites or commensals of classes (Parabasalia) and order (Preaxostyla) vertebrate hosts (Čepička et al. 2010). Table 1 identified to date, a gradual tendency towards summarises information on flagellate key diversification of the symbiotic community inside diagnostic characters (additional information on the termite hosts is evident. However, the the flagellate protists’ internal structures is also symbiotic community has been researched only in available in Table 1 of the supplementary a small number of termite species and rather material). Flagellate protists are strictly anaerobic asymmetrically, with a preponderance in some and ferment cellulose to acetate (Yamin 1980; families. Plotting the number of flagellate protist Yoshimura et al. 1996; Hongoh 2011). Acetate is groups per termite host family, it is possible to also important as a precursor for the synthesis of see an increase in diversity along host other products as amino acids or cuticular evolutionary pathway until the point at which hydrocarbons (Breznak 1982). Carbon dioxide termites completely lose their flagellate protists and hydrogen, two other products of cellulose and are able to switch to a more diversified diet fermentation, are used by the anaerobic bacteria (family Termitidae) (Fig. 3). mainly to produce acetate through reductive acetogenesis (Hongoh 2011).

Fig. 3. Simplified scheme of phylogenetic relationships within termites (based on Engel et al. 2009 and Bourguignon et al. 2015; see these authors for further details) and the number of flagellate protist species belonging to classes (Parabasalia) and order (Preaxostyla) identified to date inside termites.

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The Rhinotermitidae family is not an exception, been identified to date (C. heimi, H. indicola and and a similar pattern is observed when the C. havilandi: Yamin 1979 and references therein, subfamilies are analysed in terms of flagellate Desai et al. 2010; R. flavipes: Leidy 1877; Mello protist diversity: the basal subfamilies 1920; Cleveland 1923; Breznak & Pankratz 1977; (Rhinotermitinae, Prorhinotermitinae, Mauldin et al. 1977; Bloodgood et al. 1975; Psammotermitinae, Termitogetoninae) have three Mauldin et al. 1981; Lelis 1992; Cook & Gold or fewer flagellate protist groups, whereas 1998; Stingl & Brune 2003, Stingl et al. 2005; Coptotermitinae and Heterotermitinae, considered Brugerolle 2006, Brugerolle & Bordereau 2006, to be the sister groups of the family Termitidae Lewis & Forschler 2006; Hu 2008; Lewis & (Bourguignon et al. 2015), exhibit a higher Forschler 2010; Hu et al. 2011, Tamschick & taxonomic diversity (four to six) of flagellate Radek 2013; and references therein: Kudo 1939; protists groups. Ghidini 1942; Yamin 1979; Grassé 1982). Different types of events may have occurred However, other termite species may have a less during termite diversification, e.g., the external diverse flagellate protist community, such as uptake of symbionts or the horizontal Incisitermes snyderi (Light), with only three transmission of symbionts within different termite identified species of flagellate protists (Dolan et species which were foraging the same area and al. 2000; Gerbod et al. 2002; Harper et al. 2009). resources. The diversification of termites towards As an example of a diverse flagellate protist an optimisation of the digestive process, community, inside the European subterranean depending on the environmental conditions and termite from Portugal, Reticulitermes grassei type of resources exploited by the termites, are Clément, 12 different morphotypes were important factors to explain the flagellate protist identified based on morphological traits. The communities associated with certain groups of European subterranean termite R. grassei termites. Further insights into the evolutionary (Rhinotermitidae) is native of the Iberian pathways and constraints driving the relationships Peninsula, and also present in the Atlantic coast among termites and their symbiotic flagellate of France (Kutnik et al. 2004). One invasive protist communities are needed. This may shed population of R. grassei was identified in the UK light on the mechanisms and driving forces which (Jenkins et al. 2001), and another in Faial Island determine the establishment of symbiotic of the Azores (Ferreira et al. 2013). The flagellate relationships among different organisms and give community dynamics after an invasive event may rise to holobiotic forms of life. be of major interest for understanding the Many flagellate protists are considered species- mechanisms of adaptation to new environments specific, such as Pyrsonympha vertens Leidy and of subterranean termites. The flagellate protist Joenia annectens Grassi that have only been community from R. grassei is presumed to be identified inside the hindgut of Reticulitermes dominated by Pyrsonympha sp. (42.4%), flavipes (Kollar) and Kalotermes flavicollis Microjoenia hexamitoides Grassi (13.4%), (Fabricius), respectively. Whereas Dinenympha gracilis Leidy (10.4%), and Holomastigotes elongatum (Grassi) seems to be a Spirotrichonympha flagellata Grassi (5.4%); species with less host-specificity, as it was Trichonympha agilis Leidy (4.3%), Hexamastix identified inside 12 different termite species sp. (4.0%) and Holomastigotes elongatum Grassi belonging to three different families (3.3%) were also represented in all termites (Archotermopsidae, Hodotermitidae and observed (Duarte et al. 2016). Rhinotermitidae). However, this finding may be Flagellate protist identification errors owed to questionable as these protists’ identification relied over- or underestimation of the flagellate protist only on morphological characters evaluated by community living inside a termite hindgut are different authors (Harper et al. 2009). In some common. The misidentification of the different termite species, such as Coptotermes heimi life cycle stages of one flagellate protist species (Wasmann), Heterotermes indicola (Wasmann), as a different species is an example of Cryptotermes havilandi (Sjöstedt) or R. flavipes overestimation of flagellate protist diversity. more than 20 different flagellate protists have Underestimation errors may also occur because 31

Duarte et al. of: 1) manipulation constraints; 2) lack of & Wyman 2008). The information on the identification power of the DNA markers used; 3) identification and characterisation of endogenous the frequent concentration of research efforts on and symbiotic genes and enzymes from an the larger species, which are more easily analysed effective natural bioreactor, such as subterranean morphologically, overlooking the smaller ones, termite gut, is relevant for the improvement of and 4) misidentification of different similar industrial processes (Helle et al. 2004; Brune species as an only species through morphological 2007; Yang & Wyman 2008; Scharf & Boucias analysis (Harper et al. 2009; James et al. 2013; 2010; Tsukagoshi et al. 2014). Cellulolytic Tai et al. 2013). Nonetheless, recent advances in enzymes and genes from termites and their genomic and metagenomic techniques are leading symbiotic fauna are potential candidates for to higher quality and affordable community integrating, and consequently refining, bioethanol sequence strategies. As a consequence, production technologies, by the identification of metagenomic data on gut community of different relevant catalysts and/or by the discovery of termite host species are increasing. However, the potential recombinant enzymes which enable the lack of correspondence of operational taxonomic maximum efficiency of the processes; it is also units obtained with metagenomic analyses with possible to use mutagenesis in order to known flagellate protist species is a drawback functionally improve enzymes (e.g. Helle et al. that requires further taxonomic research efforts. A 2004; Scharf & Tartar 2008; Husseneder 2010; resilient bottleneck still remains regarding Scharf & Boucias 2010; Zhang et al. 2010; König suitable genetic markers that are taxonomically et al. 2013). and/or functionally valid and have enough Other technical processes could benefit from discrimination power regarding downstream data deep knowledge of termite and symbiont analyses. interactions inside termite gut. For example, the The diversity of flagellate protist fauna breakdown of lignocellulose into shorter associated with lower termites could be explained structural elements can form the chemical by a strong division of the labour required to building blocks for the production of new accomplish the intricate process of lignocellulose synthetic materials (Ragauskas et al. 2006). The digestion; a species or group of species acts in intestinal tract of termites is a rich source of specific phases of this process (Yoshimura et al. glycanolytic microorganisms, which may be used 1996; Inoue et al. 1997; Todaka et al. 2007; for other applications, such as the prevention of Raychoudhury et al. 2013). slime production and other undesirable side- products during vinification (Blättel et al. 2011) POTENTIAL SPIN-OFFS or for bioremediation purposes, because of their ability to degrade toxic substances (Ke et al. BIOTECHNOLOGY APPLICATIONS 2012). Furthermore, it may also be possible to use Lignocellulose is one of the most abundant termite symbionts for nitrogen fixation in soil renewable types of biomass available on earth, fertilisation (Husseneder 2010; Du et al. 2012; and cellulose-based biofuels are considered to be Thong-On et al. 2012). a sustainable alternative to reduce our dependence on fossil fuels (Ragauskas et al. 2006; Yang & NEXT-GENERATION TERMITICIDES Wyman 2008). However, the industrial Termite gut microbiota and respective cellulosic processing of lignocellulose conversion to energy activity may be a strategic target for designing needs to be adjusted in order to reduce costs and molecular-based bio-pesticides for termite control improve energy production and consequently (Zhang et al. 2010). The effectiveness of the compete with fossil fuels. For example, current potential biological control agents previously biological conversion of cellulosic biomass for studied has been compromised because of the bioethanol production is based on bacterial and symbiotic hindgut fauna, which has a protective fungal cellulolytic systems (Sun & Scharf 2010). role regarding novel and potentially harmful The most expensive part of bioethanol production microorganisms, of the termite immune system is the pretreatment (Ragauskas et al. 2006; Yang and hygienic behaviour, such as grooming

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Living inside termites activities and burying and isolation of dead heterotrophic protists in water discovered their termites (Chouvenc & Su 2012; Sun et al. 2013). important role in eliminating viruses by feeding A recent study on the synergistic effects of using on them (Deng et al. 2014). Some flagellate a nicotinoid and a pathogenic agent showed the protists inside termites may prey on bacteria by potential of this mixture to disrupt termite social ingestion (e.g. Noda et al. 2009); this process may behaviour and cause deleterious effects on the also imply eventual feeding on viruses. Possibly colonies. One of the major effects of this some protists assume the same role inside treatment was the decrease of the flagellate protist termites, and even inside other animals, acting as populations living inside the termites (Sen et al. potential elimination agents of viruses or other 2015). Knowledge on lignocellulose digestion pathogenic agents. processes may allow the definition of potential targets for novel termite control strategies based OVERVIEW on an alternative mode of action approach. The development of next-generation termiticides, Our attitude towards the described organism, targeting cellulolytic activities encoding genes, of whether for searching for more effective control endogenous or symbiont origin, with RNA strategies or for determining its correct use as a interference techniques has proven to be possible biotechnology model, should shift from the (Zhou et al. 2008; Itakura et al. 2009; Scharf et al. individual termite and its gut microbiota as 2011b; for a review of RNA interference separate entities towards a more holistic approach advances in termites and/or symbiotic protists see considering this holobiont as an independent, Scharf 2015). For example, a high-dose stranded evolutionary and functional unit. By adding an RNA force feeding trial led to the silencing of ecological and environmental axis to this two termite genes, one of them involved in holobiotic approach, we will be better able to cellulose digestion, the other in caste integrate protists’ diversity and ecology, differentiation, and this led to an increase in contributing to further applications such as: 1) mortality in the experimental population (Zhou et understanding the co-evolution mechanisms that al. 2008). lead to the establishment of this highly efficient Paratransgenesis represents a target-specific natural bioreactor and its consequent ability to strategy, which relies on the manipulation of convert lignocellulose into energy sources (Tai et genetically engineered natural symbionts (gut al. 2015); 2) the possible adjustment of diverse bacteria) which will act as a Trojan horse, as they technical industrial processes such as a are capable of surviving inside termite gut while carrying and expressing toxins which are then biorefinery (Scharf & Tartar 2008; Scharf 2015 ); spread throughout the colony by social and 3) the application of novel strategies for a interactions (Douglas 2007; Husseneder et al. more sustainable termite control in urban 2010; Rangberg et al. 2012, Sethi et al. 2014). environments (Husseneder 2010; Scharf 2015). The conjugation of this technique with a ligand- lytic peptide, which will enable the design of ACKNOWLEDGEMENTS specific ligands for flagellate protists, has already proven to be effective against lower termite pests SD’s work was supported by the Portuguese (Husseneder et al. 2010; Sethi et al. 2014). Foundation for Science and Technology (FCT) Further applications of this technology may (SFRH/BD/84920/2012). TN was supported by a involve the control of other insect pests which Marie Curie fellowship (FP7-PEOPLE-2012-CIG harbour flagellate protists and/or may act as Project Reference 321725) and by FCT vectors of protists; also, the technique could be (SFRH/BCC/52187/2013). This work was also refined to develop drugs against disease-causing supported by the project ‘Control of the Termite protists (Sethi et al. 2014). Reticulitermes flavipes in Praia da Vitória (Terceira)’ (2014-2017) funded by CMPV and IMMUNITY AGENTS ROLE AND OTHER APPLICATIONS DRA of the Azores. The authors wish to thank to Termite gut symbiotic microbiota is an active part the anonymous reviewers which contributed to the improvement of this manuscript with their of the efficient immunity system of termites comments. (Chouvenc et al. 2013). Studies on free-living

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ANNEX 1 – SUPPLEMENTARY MATERIAL

SUPPLEMENTARY TABLE 1. Description of the internal structures and body of the flagellate protists living inside lower termites and belonging to the phyla Parabasalia and Preaxostyla (order Oxymonadida). Adapted from: Honigberg et al.(1971); Brugerolle & Lee(2000); Brugerolle & Radek(2006); Čepička et al. (2010); Radek et al. (2014). Structure Description/Location pair of organelles that form the core of the centrosome Centrioles located near to the nucleus structure which function as a microtubulus organizing centre Centrosome located near to the nucleus

Basal bodies or modified centrioles that give rise to cilia and flagella kinetosomes below the cell membrane kinetosome(s) with its cytoskeletal root system Mastigont or kinetid below the cell membrane Karyomastigont mastigont plus its own associated nucleus microtubular structure which forms the cell axis, made of sheets of Axostyle microtubules spiralized, or lying in parallel layers (oxymonads), to a hollow or filled tube or cone Pelta second microtubular sheet covering the anterior end of the cell backwardly directed flagellum, running posteriorly over the body of the Recurrent flagellum (RF) flagellate in a loose or attached state; when attached it often becomes part of an undulating membrane RF adherent to a projection of the cytoplasmatic membrane (typical Undulating membrane arrangement), or the projection may also be the flagellum itself adjacent to the cytoplasmatic membrane non-microtubular striated fiber, type A or B according with the pattern of striation Costa underlain the undulating membrane specific for Parabasalia fibrillar, noncontractile structure, with subtriangular shape Cresta located below the basal portion of the trailing flagellum characteristic of devescovinid flagellates modified Golgi dictyosome Parabasal body characteristic of the Parabasalia Parabasal apparatus complex consisting of a parabasal bodies attached to striated fibers the apical end or tip of a protozoan body, when its shape is that of a beak is Rostrum some other sort of distinctive protuberance in that area of the body anterior part of the cell Flagellar apparatus basal bodies of the flagella and their connected roots any structure by which a given organism can attach, temporary or Holdfast permanently, to some living or inanimate substrate Infrakinetosomal body large, dense structure (IFK) below the basal body complex

crescent-shaped structure anterior to, and connecting, with kinetosome no.2 Suprakinetosomal body associated with IFK periodic structure, comb-shaped Comb-like structure extends between costa and IFK

43